BAZ1B regulates the PCH localization of SMARCA5 and NSD2. (A) Candidates of ISWI complexes that bind to chromatin and recruit NSD2 to PCH. (B–D) Localization of emGFP-BAZ1B and H3K36me2. iMEFs and Pmi28 cells transfected with emGFP-BAZ1B expression vector were stained with Hoechst 33342 for live-cell imaging. For immunofluorescence, cells were fixed and stained with anti-H3K36me2. (B) Examples of single optical sections for emGFP-BAZ1B (red), H3K36me2 (green), and Hoechst 33342 (blue). Contrast was adjusted individually to compare the localization patterns. (C and D) PCH-to-nucleus intensity ratios of emGFP-BAZ1B in living cells (C) and H3K36me2 by immunofluorescence (D). (E–I) Effects of BAZ1B KO on the localization of H3K36me2, NSD2, and SMARCA5. (E) BAZ1B protein levels evaluated by western blotting in WT and BAZ1B KO#9 and KO#10 iMEFs. Lamin B served as a loading control. The positions of size standards are indicated on the left. (F and G) Immunofluorescence of H3K36me2 and quantification. (F) WT, BAZ1B KO#10, and KO#9 without and with emGFP-BAZ1B were stained with anti-H3K36me2 (green) and Hoechst 33342 (red). Single optical section images are shown with individual contrast adjustments to compare the localization patterns. (G) PCH-to-nucleus intensity ratios of H3K36me2. (H and I) Intensity ratios of Halo-NSD2 (H) and Halo-SMARCA5 (I) in WT and BAZ1B KO#9 cells without and with emGFP-BAZ1B or EGFP. See Fig. 1 legend for the details of box plots. (J) Schematic drawing of NSD2 recruitment to PCH through the BAZ1B-SMARCA5 complex (WICH). Scale bars, 10 μm. Source data are available for this figure: SourceData F4.